The present invention relates to a method of measuring the degree of partitioning of a labeled species between free and bound states. The method of the present invention is especially well suited for use in competitive binding assays, such as radioimmunoassays.
In general, the method of the present invention relates to any procedure for determining the quantity or concentration of a particular material present in a liquid medium in which a labeled species is introduced into the liquid medium and subsequently is partitioned between free and bound states. As a practical matter, however, such a procedure usually is a clinical assay for minute quantities of a physiologically-or medically-significant substance in a body fluid such as blood serum or plasma. For a convenience, therefore, the discussion which follows will be limited to such clinical assays. Such discussion, however, is not to be construed as limiting either the spirit or the scope of the present invention.
Clinical assays of the type described above generally are known as competitive binding assays. Since the binding entity frequently is a protein, the term competitive protein binding assays often is used.
The principles governing the use of such assays are, of course, well known. See, for example, A. Zettner, Clin. Chem., 19, 699 (1973) and A. Zettner and P. E. Duly, Clin. Chem., 20, 5 (1974). However, a brief discussion of such principles is useful at this point to aid in understanding the present invention.
In general, competitive binding assays involve three essential components: (1) the substance, or ligand, to be measured; (2) a labeled ligand; and (3) a binding agent which is specific for the ligand and labeled ligand. During the course of the assay, both the ligand and the labeled ligand complex, at least in part, with the binding agent. Thus, the labeled ligand typically is present in both free and complexed or bound states, with the quantity of bound labeled ligand being inversely related to the initial concentration of ligand. Consequently, the quantities to be measured are the amounts of either free or bound labeled ligand, or both. In order to measure such quantities, however, it is necessary to distinguish in some manner between free labeled ligand and bound labeled ligand.
In some cases, the nature of the label employed is significantly altered upon complexation of the labeled ligand with the binding agent. This makes it possible to measure the signal emitted by the label without further processing, e.g., without separating the free labeled ligand from the bound labeled ligand. Assays making use of this phenomenon generally are referred to as homogeneous assays, the best known of which perhaps are homogeneous enzyme immunoassays employing such enzymes as lysozyme, glucose-6-phosphate dehydrogenase, and malate dehydrogenase. For a review of the current status of quantitative enzyme immunoassays, see S. L. Scharpe et al., Clin. Chem., 22, 733 (1976). See also U.K. Patent Applications GB 2,018,424-A and GB 2,018,986-A which contain descriptions of homogeneous enzyme immunoassays and homogeneous fluorescence immunoassays.
It perhaps should be noted that homogeneous assays can involve heterogeneous systems, i.e., systems consisting of a liquid phase and a solid phase. See, for example, U.K. Patent Application GB 2,019,562-A and U.S. Pat. No. 3,853,987.
In many cases, however, it is necessary to physically separate the free labeled ligand from the bound labeled ligand, particularly where the label is a radioactive element. Separation procedures are, of course, well known and include, among others, electrophoresis; electrochromatography; gel filtration; starch gel electrophoresis; equilibrium dialysis; adsorption of free labeled ligand on solid adsorbents, such as charcoal, dextran-coated charcoal, protein-coated charcoal, silica, talc, ion-exchange resins, cellulose, and cross-linked dextrans; nonspecific precipitation of proteins, including protein-bound labeled-ligand complexes, by inorganic salts, such as ammonium sulfate and sodium sulfate, or by organic solvents, such as ethanol, methanol, acetone, and dioxane; immunoprecipitation; and the like.
Most of the foregoing separation procedures require a centrifugation step to facilitate separation of the insoluble phase from the supernatant. More importantly, however, the separation step per se may be, and often is, a significant source of error. In addition, many of the procedures are time consuming and ill suited for routine use in clinical laboratories.
Most of the above-mentioned disadvantages have been eliminated or minimized with the advent of solid-phase assays. As used herein, the term "solid-phase assay" means any assay in which an essential reagent is immobilized on an insoluble support or carrier whereby the immobilized reagent forms the basis for separating free labeled ligand from bound labeled ligand. As a practical matter, the immobilized reagent typically is the binding agent, e.g., antibody specific for the ligand and labeled ligand.
Although clearly possessing numerous advantages over other prior art procedures, solid-phase competitive binding assay protocols still require multiple centrifugation and/or decantation (aspiration) steps. Where permitted by the nature of the label employed, the method of the present invention eliminates the need to physically separate the solid phase from the supernatant, thereby eliminating the need for such multiple centrifugation and/or decantation (aspiration) steps.